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(51) International Patent Classification: A62D 3/02 (2007.0 1) C08G 63/127 (2006.0 1) C08G 63/02 (2006.01) (21) International Ap

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(51) International Patent Classification: A62D 3/02 (2007.0 1) C08G 63/127 (2006.0 1) C08G 63/02 (2006.01) (21) International Ap ( 0 (51) International Patent Classification: DONOHOE, Bryon S.; c/o National Renewable Energy A62D 3/02 (2007.0 1) C08G 63/127 (2006.0 1) Laboratory, 15013 Denver West Parkway, Golden, Col¬ C08G 63/02 (2006.01) orado 80401 (US). RORRER, Nicholas; c/o National Re¬ newable Energy Laboratory, 15013 Denver West Parkway, (21) International Application Number: Golden, Colorado 80401 (US). MCGEEHAN, John E.; PCT/US20 19/0 19502 c/o University of Portsmouth, University House, Winston (22) International Filing Date: Churchill Ave., Portsmouth Hampshire P01 2UP (GB). 26 February 2019 (26.02.2019) AUSTIN, Harry P.; c/o University of Portsmouth, Uni¬ versity House, Winston Churchill Ave., Portsmouth Hamp¬ (25) Filing Language: English shire P01 2UP (GB). ALLEN, Mark D.; c/o University (26) Publication Language: English of Portsmouth, University House, Winston Churchill Ave., Portsmouth Hampshire P01 2UP (GB). (30) Priority Data: 62/636,594 28 February 2018 (28.02.2018) US (74) Agent: HALL, Alexandra M.; Alliance for Sustainable Energy, LLC, c/o National Renewable Energy Laborato¬ (71) Applicants: ALLIANCE FOR SUSTAINABLE EN¬ ry, 15013 Denver West Parkway, Golden, Colorado 80401 ERGY, LLC [US/US]; c/o National Renewable Energy (US). Laboratory, 15013 Denver West Parkway, Golden, Col¬ orado 80401 (US) UNIVERSITY OF PORTSMOUTH (81) Designated States (unless otherwise indicated, for every [GB/GB]; University Flouse, Winston Churchill Ave., kind of national protection av ailable) . AE, AG, AL, AM, Portsmouth Flampshire P01 2UP (GB). AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, (72) Inventors: BECKHAM, Gregg Tyler; c/o National Re¬ DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, newable Energy Laboratory, 15013 Denver West Parkway, HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, Golden, Colorado 80401 (US). JOHNSON, Christopher KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, W.; c/o National Renewable Energy Laboratory, 15013 MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, Denver West Parkway, Golden, Colorado 80401 (US). OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (54) Title: ENZYMES FOR POLYMER DEGRADATION (57) Abstract: Disclosed herein are engineered enzymes capable of degrading polymers such as polyethylene terephthalate (PET). Also disclosed are nucleic A acids encoding the engineered enzymes and cells that express the engineered ing polymers such as aromatic and semi-aromatic [Continued on next page] ENZYMES FOR POLYMER DEGRADATION CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62/636,594 filed on February 28, 2018, the contents of which are incorporated herein by reference in their entirety. CONTRACTUAL ORIGIN The United States Government has rights in this invention under Contract No. DE- AC36-08GO28308 between the United States Department of Energy and Alliance for Sustainable Energy, LLC, the Manager and Operator of the National Renewable Energy Laboratory. REFERENCE TO SEQUENCE LISTING This application contains a Sequence Listing submitted as an electronic text file entitled “NREL 18-54 Sequence Listing_ST25.txt,” having a size in bytes of 8 kb and created on February 6, 2019 Pursuant to 37 C.F.R. § 1.52(e)(5), the information contained in the above electronic file is hereby incorporated by reference in its entirety BACKGROUND In less than a century of manufacturing, plastics have become essential to modern society, driven by their incredible versatility coupled to low production costs. It is, however, now widely recognized that plastics pose a dire global pollution threat, especially to marine wildlife and ecosystems, because of the ultra-long lifetimes of most synthetic plastics in the environment. In response to the accumulation of plastics in the biosphere, it is becoming increasingly recognized that microbes are adapting and evolving enzymes and catabolic pathways to partially degrade man-made plastics as carbon and energy sources. These evolutionary footholds offer promising starting points for industrial biotechnology and synthetic biology to help address the looming environmental threat posed by man-made synthetic plastics. Polyethylene terephthalate (PET) is the most abundant polyester plastic manufactured in the world. Most applications that employ PET, such as single-use beverage bottles, clothing, packaging, and carpeting, employ crystalline PET, which is recalcitrant to catalytic or biological depolymerization due to the limited accessibility of the ester linkages. In an industrial context, PET can be depolymerized to its constituents via chemistries able to cleave ester bonds. However, to date, few chemical recycling solutions have been deployed given the high processing costs relative to the purchase of inexpensive virgin PET. This in turn results in reclaimed PET primarily being mechanically recycled, ultimately resulting in a loss of material properties, and hence intrinsic value. Given the recalcitrance of PET, the fraction of this plastic stream that is landfilled or makes its way to the environment is projected to persist for hundreds of years. The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings. The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools, and methods that are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other improvements. Exemplary embodiments provide a modified poiytethylene terephthalate) (PET)- digesting enzyme (PETase) that exhibits improved polymer degradation capacity relative to wild-type PETase due to its narrowed binding cleft via mutation of two active-site residues is disclosed. In some embodiments, unmodified PETase is from a bacterium of the genus Ideonella. In some embodiments, the bacterium is a strain of Ideonella sakaiensis. In some embodiments, an amino residue at position 159 is mutated. In some embodiments, an amino residue at position 238 is mutated. In some embodiments, an amino residue at position 238 is mutated. In some embodiments, the modified PETases comprises the W159H/S238F double mutation. In exemplary embodiments, a nucleic acid molecule encoding a modified PETase having mutations at two active-site residues is disclosed. In some embodiments, the amino acid residue at position 159 is mutated. In some embodiments, the amino acid residue at position 238 is mutated. In some embodiments, the modified PETase comprises the W159H/S238F double mutation. In exemplary embodiments, an expression vector comprising the nucleic acid molecule encoding a modified PETase having mutations at two active-site residues is disclosed. Exemplary embodiments provide a nucleic acid encoding the enzyme comprising the amino acid sequence depicted in FIG. 2(B). In others, the cell that expresses the modified poly(ethylene terephthalate) (PET)-digesting enzyme (PETase) that exhibits improved polymer degradation capacity relative to wild-type PETase due to its narrowed binding cleft via mutation of two active-site residues. Exemplary embodiments provide method for degrading a polymer comprising contacting the modified PETase of claim 1 or the cell of claim 3 with the polymer. In some methods, the polymer is a polyester. In other methods, the polymer is an aromatic polymer or a semi-aromatic polymer. In some methods, the polymer is polyethylene terephtha!ate (PET). In other methods, the polymer is polyethylenefuranoate (PEF). In certain methods the polymer is from a recycled plastic material. In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following descriptions. BRIEF DESCRIPTION OF THE DRAWINGS Exemplary embodiments are illustrated in referenced figures of the drawings. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than limiting. FIG. shows the nucleotide (A) and amino acid (B) sequences of PETase from Ideonel a sakaiensis. FIG. 2 shows the nucleotide (A) and amino acid (B) sequences of PETase from Ideonella sakaiensis containing the W159H and S238F mutations (bold and underlined). FIG. 3 shows high resolution X-ray crystallography data collection and analysis of PETase. FIG. 4 illustrates the structure of PETase. FIG. 5 shows PETase sequence analysis. FIG. 6 illustrates the compari so of the active site cleft of PETase with utinases FIG. 7 show's multiple sequence alignments of PETase with lipase and cutinase family members. FIG. 8 illustrates the structural and functional analysis of key residues in PETase. FIG. 9 shows the chemical analysis of polymer substrates. FIG. 0 illustrates a comparison of PETase and the engineered enzyme S238F/W159H with PET. FIG. shows the induced fit docking analysis of PETase and the engineered enzyme S238F/W1 59H with PET. FIG. 12 show's degradation analysis of PBS and PLA by PETase. FIG. 3 illustrates a comparison of PETase and the engineered enzyme S238F/W159H with PEF. DETAILED DESCRIPTION Disclosed herein are engineered enzymes capable of degrading polymers and plastics such as polyethylene terephthalate (PET). Such enzymes include PET-degrading enzymes (PETases) wherein certain amino acid residues are mutated to different amino acids to improve enzymatic activity. One example of an engineered PETase as disclosed herein is provided in FIG. 2, which provides nucleotide and amino acid sequences for a PETase from the bacterium Ideonella sakaiensis wherein the tryptophan residue at position 59 has been mutated to a histidine residue and the serine residue at position 238 has been mutated to a phenylalanine (W159H/S238F). Ideonella sakaiensis 201-F6 is a bacterial strain with the ability to use PET as its major carbon and energy source for growth.
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